The Cryptococcus genus spans a diverse group of fungal species from environmental saprophytes to common human pathogens. In fact, the Cryptococcus neoformans and Cryptococcus gattii species complexes cause >220,000 life-threatening infections each year in both immunocompromised and immunocompetent patients, leading to >180,000 deaths, >15% of all HIV/AIDS-related deaths, and >70% mortality in low-income countries. In studies supported by this award, we defined the structure, function, and evolution of a large genomic region in Cryptococcus species that has co-evolved with pathogenesis, the fungal mating type (MAT) locus. The Cryptococcus MAT locus is a large, complex gene cluster that controls sexual reproduction, infectious spore production, and pathogenicity. We elucidated how these sex- and virulence-determining genes evolved from a nonpathogenic ancestral state. Our studies reveal bipolar/inbreeding mating systems are shared by all of the pathogenic species, and evolved from an ancestral outbreeding/tetrapolar system. Similar transitions occurred in other fungal pathogens of plants and animals, suggesting convergent evolution in concert with host adaptation. In the prior award period, we made major advances characterizing the association of fungal MAT locus evolution with virulence: 1) we sequenced the genomes/MAT loci of 24 species spanning all Cryptococcus pathogens and aligned nonpathogens; 2) we defined Cryptococcus centromeres and implicated inter- centromeric recombination in mating type and genome transitions; 3) we demonstrated the MAT-encoded RPL22 genes have essential, specialized functions. In the current proposal, we hypothesize that mating-type transitions drove pathogen emergence resulting in a gene complex linked to virulence and infectious spore production. Our recent studies reveal unique aspects of MAT biology allowing us to propose new aims to test this hypothesis. Aim 1 focuses on MAT locus structure and evolution, utilizing RNA-Seq and sRNA analysis to define mRNA, asRNA, lncRNA, and siRNA produced by MAT during asexual/sexual reproduction, engineering translocations to model tetrapolar-bipolar transition with CRISPR, and conducting Hi-C analysis to examine nuclear organization of MAT and centromeres. Aim 2 will elucidate MAT functions linked to virulence and development involving 1) MAT-encoded lncRNA ASM1, 2) diverged, specialized, essential ribosomal genes we hypothesize operate as an ancestral imprinting system ensuring sexual reproduction fidelity, and 3) modeling in mice of the role of a newly recognized host factor controlling immune protection against cryptococcal infections: host GM-CSF signaling. Auto-antibodies against the GM-CSF cytokine are a major risk factor for lineage-specific Cryptococcus infections, suggesting unique host features might play a role in evolution of infections due to human pathogenic species. These studies will advance understanding of dynamic microbial genome evolution, associating specific gene clusters to virulence and revealing unique host susceptibility determinants, with direct implications for infectious disease evolution, treatment, and prevention.